ArticleBMC neuroscience2022
Impacts of autofluorescence on fluorescence based techniques to study microglia.
Article in BMC neuroscience, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 21 citations in OpenAlex.
- High-Content Immunofluorescence Quantification of Transcription Factors: Application to pSMADs for BMP/TGFβ Signaling Pathways.Current protocols · 2026Article
- Exploring the human brain: spatial transcriptomics challenges and approaches in post-mortem analysis.Brain : a journal of neurology · 2026Review
- Microglial Autofluorescence in the Brain and Retina is Dynamically Modulated by Systemic Inflammation.Cellular and molecular neurobiology · 2026Article
- Differential pericyte pathology in the human retina and brain in diabetes mellitus and Alzheimer's disease.Frontiers in neuroscience · 2026Article
- Meningeal lymphatic architecture and drainage dynamics surrounding the human middle meningeal artery.iScience · 2025Article
- Multiparameter quantitative analyses of diagnostic cells in brain tissues from tuberous sclerosis complex.Cytometry. Part B, Clinical cytometry · 2025Article
- Feasibility of Nanostructured Lipid Carrier Loaded with Alpha-Mangostin and Clove Oil for Canine Periodontal Therapy.Animals : an open access journal from MDPI · 2024Article
- Synthesis and Characterization of Click Chemical Probes for Single-Cell Resolution Detection of Epichaperomes in Neurodegenerative Disorders.Biomedicines · 2024Article
- Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment.Nature communications · 2023Article
- Volumetric live-cell autofluorescence imaging using Fourier light-field microscopy.Biomedical optics express · 2023Article
- Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy.Bioengineering (Basel, Switzerland) · 2023Article
- Label-free macrophage phenotype classification using machine learning methods.Scientific reports · 2023Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundMicroglia, the resident immune cells in the central nervous system, accrue autofluorescent granules inside their cytoplasm throughout their lifespan. In this report, we studied the impacts of autofluorescence on widely used fluorescence-based techniques to study microglia, including flow cytometry, immunofluorescence staining, and live imaging.
resultsThe failed attempt of using fluorescein isothiocyanate (FITC) conjugated antibody to detect lymphocyte-activation gene 3 protein in microglia prompted us to compare the sensitivity of FITC, phycoerythrin (PE) and allophycocyanin (APC) conjugated antibodies to detect surface protein expression in microglia. We found that PE outperformed FITC and APC as the fluorophore conjugated to antibody for flow cytometry by overcoming the interference from microglia autofluorescence. To identify the location and source of microglia autofluorescence, we did confocal imaging and spectral analysis of microglia autofluorescence on fixed brain tissues, revealing that microglia autofluorescence emitted from cytoplasmic granules and displayed a multi-peak emission spectrum. We recommended removing autofluorescence by lipofuscin removing agents when staining intracellular proteins in microglia with the immunofluorescence techniques. On live brain slices, autofluorescent granules reduced the amplitudes of calcium signals in microglial somata derived from GCaMP6s fluorescence and thus needed to be excluded when selecting regions of interest (ROI).
conclusionsIn conclusion, autofluorescence is a critical factor to consider when designing experiments and interpreting results based on fluorescence-based techniques to study microglia.
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